Prophylaxis of disease caused by bacterial pathogens of man

This thesis reports research undertaken which will lead to improved pretreatments and therapies for disease caused by Clostridium perfringens, Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. C. perfringens is thought to be the most widely distributed bacterial pathogen and is the most important Clostridial species associated with enteric disease in domesticated animals. During warfare C. perfringens has been a significant causes of mortality. Between 1 and 10% of wounded personnel developed gas gangrene during the 1st and 2nd world wars. The ability of the bacterium to cause a range of diseases is due largely to the differential production of toxins. The first reported cloning and nucleotide sequencing of three of the four major toxins (α, β and ε-toxins) is documented in this thesis. The regulation of expression of α-toxin in C. perfringens has been investigated and methods for the expression of recombinant proteins in E. coli have been devised This information has been used to develop improved PCR-based diagnostic tests, and to investigate structure-function relationships. A high resolution crystal structure of a-toxin (phospholipase C) is reported. Using molecular and biophysical techniques, the functions of the two domains of the protein have been determined. Residues that play roles in the interaction of the toxin with host cell membranes have been identified using site-directed mutagenesis. This work has also provided a major insight into the structures and functions of related phospholipases C (the zincmetallophospholipases C) from other bacterial pathogens. This pioneering work with α-toxin is recognised by invitations to write reviews and book chapters on this subject and on bacterial phospholipases C. C. perfringens β-toxin has been shown to be related to pore forming toxins such as Staphylococcus aureus α-toxin. This finding suggests, for the first time, the mode of action of β-toxin. The interaction of C. perfringens ε-toxin with host cells has been investigated and progress made in identifying the cell-surface receptor for the toxin. Genetically engineered toxoids have been devised which induce high-level protection against α and ε-toxins. These vaccines are currently being developed by industry for veterinary use. Similar approaches have been used to devise a recombinant vaccine against Clostridium botulinum toxin F. The wider applications of toxins as therapeutics have also been investigated, and a novel cancer drug delivery system based on targeted lysis of drug-containing liposomes by α-toxin has been devised and patented. F. tularensis is the etiological agent of tularemia, a disease of man that is found in most countries in the Northern hemisphere and most frequently in Scandinavia, N. America, Japan and N. Russia. In this thesis the efficacy of antibiotics for the prevention and treatment of experimental tularemia is documented. Two surface antigens (lipopolysaccharide and FopA) have been evaluated as sub-unit vaccines. Of these, lipopolysaccharide shows potential as a protective antigen. However, because of the paucity of information available on this bacterium, a wider approach to vaccine development, involving the determination of the genome sequence of a fully virulent strain of F. tularensis has been undertaken. A preliminary analysis of the genome sequence is reported here, which has allowed the identification of targets for the development of a rationally attenuated mutant for use as a live vaccine. Y. pestis is generally recognised to have caused three major pandemics of disease, and credible estimates indicate that together these resulted in 200 million deaths. WHO figures indicate that there is a continuing public health problem from plague, especially in Africa, Asia and South America. In this thesis existing vaccines and antibiotics have been evaluated for the prevention and treatment of plague and found to have limitations. A number of approaches to the development of an improved vaccine have been investigated including rationally attenuated strains of the bacterium and isolated surface antigens. A sub-unit vaccine against plague has been devised based on recombinant forms of the F1- and V-antigens. This vaccine provides high level protection against both bubonic and pneumonic plague. This recombinant sub-unit vaccine has been patented and is currently in phase I clinical trials in man. This vaccine has been formulated for single oral or intranasal delivery, using microencapsulated or Salmonella-based delivery systems. Methods for enhancing the stability and efficacy of these vaccines have been investigated. Reviews on plague and plague vaccines have been written, confirming the status of the author as a world leader in this field. The work to devise an improved vaccine has also provided insight into the molecular basis of pathogencity of Y. pestis. A phoP / phoQ regulatory system has been discovered in the bacterium, which plays a key role in survival of the bacterium within macrophages. The V-antigen has been shown to be surface located to play a key role in the translocation of effector proteins into host cells. The biogenesis of the F1-capsular antigen has been investigated at a genetic and biophysical level. In order to underpin future work with this pathogen, the genome sequence is currently being determined. This work has already provided major new insights into the evolution of this pathogen. B. pseudomallei (formerly Pseudomonas pseudomallei) is found primarily in S. E. Asia, N. Australia and other tropical areas of the world. Melioidosis has recently appeared in temperate zones, including mainland France and the UK possible as a consequence of increased international travel. Acute disease can be treated with antibiotics but the bacterium can persist in the host and subsequent disease episodes can occur. In this thesis ciprofloxacin and doxycyline have been are evaluated and shown to have significant limitations for the treatment of melioidodis. In the longer term there is a requirement for an effective vaccine against melioidosis, and work is reported here to devise the genetic tools which will be necessary for the genetic manipulation of the bacterium, with a view towards the identification of virulence determinants

Characterization and Deposition of Respirable Large- and Small-Particle Bioaerosols ▿

The deposition patterns of large-particle microbiological aerosols within the respiratory tract are not well characterized. A novel system (the flow-focusing aerosol generator [FFAG]) which enables the generation of large (>10-μm) aerosol particles containing microorganisms under laboratory conditions was characterized to permit determination of deposition profiles within the murine respiratory tract. Unlike other systems for generating large aerosol particles, the FFAG is compatible with microbiological containment and the inhalational challenge of animals. By use of entrapped Escherichia coli cells, Bacillus atrophaeus spores, or FluoSphere beads, the properties of aerosols generated by the FFAG were compared with the properties of aerosols generated using the commonly available Collison nebulizer, which preferentially generates small (1- to 3-μm) aerosol particles. More entrapped particulates (15.9- to 19.2-fold) were incorporated into 9- to 17-μm particles generated by the FFAG than by the Collison nebulizer. The 1- to 3-μm particles generated by the Collison nebulizer were more likely to contain a particulate than those generated by the FFAG. E. coli cells aerosolized using the FFAG survived better than those aerosolized using the Collison nebulizer. Aerosols generated by the Collison nebulizer and the FFAG preferentially deposited in the lungs and nasal passages of the murine respiratory tract, respectively. However, significant deposition of material also occurred in the gastrointestinal tract after inhalation of both the small (89.7%)- and large (61.5%)-particle aerosols. The aerosols generated by the Collison nebulizer and the FFAG differ with respect to mass distribution, distribution of the entrapped particulates, bacterial survival, and deposition within the murine respiratory tract

The transfer of a laboratory based hypothesis to a clinically useful therapy: the development of anti-TNF therapy of rheumatoid arthritis.

The development of anti-TNF therapy is a key step forward in rheumatology as it is the first new therapy for based on investigating the molecular mechanisms of this disease. This chapter reviews how this discovery was made

Absence of Yersinia pestis-specific DNA in human teeth from five European excavations of putative plague victims

© 2004 Society for General MicrobiologyThis study reports the results of a collaborative study undertaken by two independent research groups to (a) confirm recent PCR-based detection of Yersinia pestis DNA in human teeth from medieval plague victims in France, and (b) to extend these observations over five different European burial sites believed to contain plague victims dating from the late 13th to 17th centuries. Several different sets of primers were used, including those previously documented to yield positive results on ancient DNA extracts. No Y. pestis DNA could be amplified from DNA extracted from 108 teeth belonging to 61 individuals, despite the amplification of numerous other bacterial DNA sequences. Several methods of extracting dentine prior to the DNA extraction were also compared. PCR for bacterial 16S rDNA indicated the presence of multiple bacterial species in 23 out of 27 teeth DNA extracts where dentine was extracted using previously described methods. In comparison, positive results were obtained from only five out of 44 teeth DNA extracts for which a novel contamination-minimizing embedding technique was used. Therefore, high levels of environmental bacterial DNA are present in DNA extracts where previously described methods of tooth manipulation are used. To conclude, the absence of Y. pestis-specific DNA in an exhaustive search using specimens from multiple putative European plague burial sites does not allow us to confirm the identification of Y. pestis as the aetiological agent of the Black Death and subsequent plagues. In addition, the utility of the published tooth-based ancient DNA technique used to diagnose fatal bacteraemias in historical epidemics still awaits independent corroboration.M. Thomas P. Gilbert, Jon Cuccui, William White, Niels Lynnerup, Richard W. Titball, Alan Cooper and Michael B. Prentic

Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique “genomic islands” identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen